The principal objectives of this proposal are to elucidate and characterize selected physicochemical and functional properties of the endoplasmic reticulum (E.R.) in hepatocytes. The series of interrelated projects will apply biochemical/biophysical rationale, and will have broad implications in terms of hepatic transport physiology and membrane/enzyme biology. Specifically, the role of membranes as a medium for the intracellular transport of lipophilic substrates and bile salts will be studied using native E.R. and model membranes, and an ultra-rapid filtration system. The mechanism(s) of membrane-membrane transfer and the influence of substrate hydrophilicity, cytosolic binding proteins, and phospholipid transfer proteins will be examined, as well as the impact on rates of microsomal biotransformation. Since bile salt synthesis and metabolism are intimately related to the E.R., the mechanisms and regulation of radiolabeled and fluorescent bile salt uptake by microsomal membranes will be defined, and the concept that bile salts self-associate to form reverse micelles in native membranes and thereby function as diffusional membrane carriers for polar molecules will be investigated. The effects of a physiologic series of bile salts on the composition, structure and physical state of E.R. and Golgi membranes will be studied using physicochemical techniques, including fluorescence polarization, differential scanning calorimetry and quasi- elastic light scattering. We also will examine the postulates that bile salts regulate the activity of microsomal enzymes (i.e. UDP- glucuronosyltransferase (GT) and cytochrome P-450), and provide the driving force for biliary lipid excretion, by initiating microvesiculation of the E.R., with formation of lipid particles (vesicles) that undergo vectorial transport to the canalicular membrane. Detailed physicochemical analysis (e.g. phospholipid species, static/dynamic fluidity) of E.R. membranes will be employed to delineate whether structural/compositional modifications of microsomal enzymes. Finally, using a rapid filtration assay, we will investigate and characterize the functional significance, regulation and properties of the membrane carrier, which transports UDP-glucuronic acid (the essential cosubstrate for all glucuronidation reactions) from the cytosol into the E.R. Collectively, these findings will enhance our understanding of the diverse and pivotal functions of the endoplasmic reticulum in hepatocytes, and will have implications with regard to intracellular vectorial and transmembrane transport processes, the regulation and properties of membrane-bound enzymes, and the subcellular effects, detoxication and biliary excretion of an array of lipophilic endogenous and xenobiotic substrates.